Expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) or Oct4, Sox2, Nanog, and Lin-28 in fibroblasts can "reprogram" them to become induced pluripotent stem (iPS) cells, which are highly similar to embryonic stem (ES) cells. iPS cells will allow researchers to create model systems to study complex genetic diseases and potentially to create patient-specific therapies. Reprogramming occurs with extremely low efficiency, and the mechanism is poorly understood. Recently, Lin-28 was found to inhibit processing of the let-7 microRNA (miRNA) family in ES cells. Our preliminary data indicate that adding Lin-28 to OSKM increases reprogramming efficiency. We propose that Lin-28's role in direct reprogramming is by inhibiting the processing of specific miRNAs. Specific Aim 1. To identify miRNAs that are downstream effectors of Lin-28 during reprogramming. We will express Lin-28 in fibroblasts and knockdown Lin-28 in ES cells to 1) determine which of the let-7 family members are most affected by Lin-28 expression by RT-PCR and 2) identify novel miRNAs regulated by Lin-28 by using miRNA microarrays. Specific Aim 2. To determine if the miRNAs regulated by Lin-28 contribute to the positive effect of Lin-28 on reprogramming. We will express OSKM and knockdown miRNAs using miRNA inhibitors or sponges for the miRNAs identified in Aim 1 and count the number of colonies obtained to determine if lack of particular miRNAs increases reprogramming efficiency. Specific Aim 3. To identify targets of the miRNAs that effect reprogramming. We will identify genes targeted by the miRNAs found to inhibit reprogramming in Aim 2 and infect fibroblasts with these target genes in combination with OSKM to determine if these genes either enhance or inhibit reprogramming. Our study will provide insight into how skin cells can become iPS cells. We hope that this work will also allow us to create IPS cells with small molecules rather than inserting genes into the skin cell DNA, thus alleviating the cancer risk with using iPS cells for therapy. As a skin cell converting into a pluripotent cell may be similar to a normal cell becoming a cancer cell, our study of iPS cells may lead to insights in the field of cancer cell biology and new targets for developing cancer therapies.